Faulty DNA repair can lead to increased mutations, formation of cancers, and cell death. The process by which repair proteins find damaged bases within the DNA represents an important type of protein-DNA interaction, which is not well-understood. The UvrA, UvrB,and UvrC proteins work together to identify and remove DNA damage in a process called nucleotide excision repair. One of the most remarkable aspects of NER is that it can remove a wide range of DNA lesions that differ in chemistry and structure. The UvrABC proteins are believed to recognize the damage-induced distortion in the DNA helix rather than the lesion per se. However, detailed studies of the kinetics,thermodynamics and structural aspects of the Uvr proteins have been limited due to the lability and instability of the proteins. To overcome this problem we have recently cloned and overexpressed UvrA, UvrB,and UvrC from the thermophile, Bacillus caldotenax. The proteins maintain their activity at 65oC and are more amenable to structural and biophysical studies. Work is underway to understand the structure and function of these proteins using x-ray crystallography, stopped-flow fluorescence and site-directed mutagenesis. Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism present in all kingdoms of life. UvrB is a central component of the bacterial NER system, participating in damage reognition, strand excision and repair synthesis. None of the three presently available crystal structures of UvrB has defined the structure of domain 2, which is critical for the interaction with UvrA. We have solved the crystal structure of the UvrB Y96A variant, which reveals a new fold for domain 2 and identifies highly conserved residues located on its surface. These residues are restricted to the face of UvrB important for DNA binding and may be critical for the interaction of UvrB with UvrA. We have mutated these residues to study their role in the incision reaction, formation of the preincision complex, destabilization of short duplex regions in DNA, binding to UvrA and ATP hydrolysis. Based on the structural and biochemical data, we conclude that domain 2 is required for a productive UvrA?UvrB interaction, which is a pre-requisite for all subsequent steps in nucleotide excision repair. Future work is focused at understanding the formation of the repair complexes on DNA using Atomic Force microscopy. These studies have revealed that UvrB might act as a dimer.